Lysis of Viable Yeast Cells by Enzymes of Arthrobacter Luteus

Abstract

An enzyme which lysed viable yeast cells was purified about 75-fold from the culture fluid of Arthrobacter luteus by procedures including salting out with ammonium sulfate and Biogel CM-100 column chromatography. The purified enzyme appeared homogeneous on electrophoresis and ultracentrifugation, and had a molecular weight of about 21,000. This enzyme lysed viable yeast cells in the absence of any other enzymes or additives and was tentatively named zymolyase. The optimum pH for lysis of viable yeast cells was 7.0-7.5, the optimum temperature was 35°, and the enzyme was relatively stable at pH 5-11. About 70 % of its activity was lost on incubation at 50° for 5 min, and all its activity was lost on incubation at 60° for 5 min. Studies on the hydrolyses of j3-1,3-glucans, i.e., yeast glucan, pachyman, curdlan, laminarin, and laminaran, showed that zymolyase is an endo-jS-1,3-glucanase, which specifically releases laminaripentaose as the minimum product unit. 1-lowever, it behaves like an exo-enzyme in hydrolysis of high molecular jS-1,3-glucan, which shows strong molecular aggregation like yeast glucan, releasing laminaripentaose units. Zymolyase shows much more affinity for insoluble glucan than for soluble glucan. Previously we reported that the culture broth of a strain of Arthrobacter luteus accumulated lytic activity for viable yeast cells only when the culture medium contained j3-1,3-glucan, and that the pattern of accumulation of this activity was quite different from that of-1,3-glucanase (EC 3.2.1.6) (1). These differences 323 324 KTTAMURA, KANEKO, and YAMAMOTO VOL. 20 suggested that some essential factors, other than fi-1,3-glucanase, might be involved in the lyses of viable yeast cells. This paper describes the purification, properties, and mode of action of an enzyme of Arthrobacter luteus, which lyses viable yeast cells but differs from fl-1, 3-glucanase (EC 3.2.1.6). MATERIALS AND METHODS Strain, medium, cultivation and crude preparation of the yeast-lytic enzyme. Arthrobacter luteus B 111-1 was cultivated in dried yeast medium (dried yeast 2 %, yeast extract 0.2 %, NH4NO3 0.2 %, K2HPO4 0.1 %, MgSO4.7H2O 0.1 %, and Fe2(S04)3 • xH2O 0.01 %, pH 10) at 30° for 24 hr with aeration. The culture broth was centrifuged at 8,000 rpm for 10 min. The crude enzyme preparation was obtained from the supernatant by the same method as described in a previous report (1). fl-1,3-Glucanase. Endo-fl-1 ,3-glucanase was prepared by gel filtration on Sephadex G-50 from Glucanase G-4, kindly provided by Dr. Y. Takagi, Amano Seiyaku Co., Ltd. Carbohydrates. Yeast glucan was prepared from brewery yeast by the method of PEAT et al. (2). Pachyman was prepared from Poria cocos as follows : The powdered fungus was added to 0.1 N NaOH solution to 0.5 % concentration, the mixture was stirred for 30 min at room temperature, and then filtered. Pachyman was precipitated from the filtrate by neutralization with 0.2 N HCI. After being washed twice with water, the precipitate was dissolved in 0.1 N NaOH solution and centrifuged. Pachyman...

Heteronuclear NMR studies of 13C-labeled yeast cell wall ?-glucan oligosaccharides

“…Cell walls were then washed with H20 and lyophilized (360 mg dry weight). Walls suspended in 45 ml 0.01 M Na/K phosphate buffer, pH 7.5, were then digested at 37 ~ for 15 h with laminarapentaohydrolase (388 p.g, 0.4 A280 units) prepared from Zymolyase 100T (Seikagaku Inc., Rockville, MD) as described previously (Kitamura et al, 1974). The digest was heated to 65 ~ for 10 min to inactivate the enzyme, centrifuged at 10 000 x g for 10 min, and then the supernatant was Abbreviations."…”

Lysis of Yeast Cells Showing Low Susceptibility to Zymolyase

“…The purified preparation was obtained as described in a previous report. 3 ) Yeast. Baker's yeast and Saccharomyces cerevisiae IFO 0283 were used.…”

“…The enzyme unit was determined as described in the previous report. 3 ) Determination of fJ-I,3-glucanase activity. The reaction mixture containing I ml of heat-treated pachyman suspension (2.5 mg/ml),3) 2.5 ml of 0.05 M Tris buffer (pH 7.5), 1 ml of water and 0.5 ml of enzyme solution, was incubated at 45°C for 30 min.…”

The Synergistic Effects amongβ-,3-Glucanases fromOerskoviasp. CK on Lysis of Viable Yeast Cells

“…The lysis of viable yeast cells has been reported with fJ-l,3-g1ucanases of Arthrobacter, 4 2 ,31 However, the complex structure of the yeast cell wall consisted of glucan, mannan, protein and chitin components has prevented clear elucidation of the mechanism of the enzymatic lysis.…”